Composition comprising notoginseng radix extract for preventing and treating of arthritis as an effective ingredient

ABSTRACT

The present invention relates to a composition comprising  Notoginseng radix  extract for preventing and treating arthritis as an effective ingredient.  
       Notoginseng radix  extract of the present invention inhibits release of tumor necrosis factor-alpha (TNF-α) and is the death of activated T-cells only, so that it can be effectively used for the production of a medicine for preventing and treating arthritis and health food as well.

TECHNICAL FIELD

The present invention relates to a composition comprising Notoginsengradix extract for preventing and treating arthritis as an effectiveingredient.

BACKGROUND ART

Arthritis related diseases are the representative degenerativeintractable diseases, which give 12% of total earth population pain. Andover 2 million people are suffering from such diseases in Korea.

Arthritis is the general term for symptoms over all the musculoskeletalsystem caused by inflammatory changes in musculoskeletal and connectivetissues. The disease is characterized by chronic inflammation causingpermanent damage in tissues, deformity, degeneration and troubles byhaving an effect on joint, bone, cartilage or the spinal cord (Hofbause,L C, Heufelder, A E: The role of osteoprotegerin and receptor activatorof nuclear factor kappaB ligand in the pathogenesis and treatment ofrheumatoid arthritis, Arthritis and Rheumatism 44:253-259, 2001).

Arthritis is classified into degenerative arthritis (osteoarthritis),rheumatoid arthritis, non-joint rheumatism or collagen disease.

Degenerative arthritis, which is the most common of all arthritisrelated diseases, is developed by local degeneration by the worn-out ofjoint cartilage. The cause of the disease is still unclear but aging orover-weight might be the reason. Primarily, degenerative changes appearin joint cartilage. Degeneration first begins in joint cartilage andkills chondrocytes and then cartilage matrix is destroyed by cathepsinB, cathepsin D, collagenase, etc. The destruction outpaces thegeneration of proteoglycan and collagen, and adaptability of cartilageto outside force becomes weaker, resulting in microfractures insubchondral bone tissues. As the disease progresses, the hardening ofsubchondral bone, over-ossification around joint, joint deformation,etc. are observed. Then, the surface of cartilage becomes rough andinflammation in joint cavity enveloped by joint capsule repeats,resulting in constant pain, ankylosis and gradual motor disturbance injoint.

Rheumatoid arthritis is a chronic inflammatory disease over the wholebody and its symptoms occur symmetrically to movable joints. The diseaseis also known as an autoimmune disease caused by malfunction of immunesystem. However, the cause of the disease is still in question.Rheumatoid arthritis is characterized by continuous inflammatorysynovitis causing the destruction of cartilage and bone erosion,resulting in deformity of joint structure. Symptoms of rheumatoidarthritis are joint edema, joint tenderness, inflammation, morningstiffness and acute pain with bending. As the disease progresses,structural damage can be found such as bone erosion and jointdestruction (Firestein, G S: Evolving concept of rheumatoid arthritis.Nature 423:356-361, 2003). In addition, a patient with rheumatoidarthritis might suffer from other symptoms by additional organ damage,for example damage of skin, kidney, heart, lung, central nervous systemand eye, which is resulted from vasculitis related to autoimmuneprocess.

Arthritis related symptoms include acceleration of erythrocytesedimentation rate and increase of the concentration of serum C-reactiveprotein (CRP) or soluble IL-2 receptor (IL-2r). The acceleration oferythrocyte sedimentation rate is detected in almost every activerheumatoid arthritis patients. The concentration of serum C-reactiveprotein also increases in those patients. It is related to theactivation of the disease and the possibility of progressive jointdamage. The concentration of soluble IL-2r, a product of T-cellactivation, increases in serum and synovial fluid of active rheumatoidarthritis patients, too (Udagawa, N., Kotake, S., Kamatani, N.,Takahashi, N., and Suda, T: The molecular mechanism ofosteoclastogenesis in rheumatoid arthritis. Arthritis Research4:281-289, 2002).

It is generally believed that Th1 type CD4+ T cells play an importantrole in the progress and continuation of rheumatoid arthritis. That is,CD4+ T lymphocytes stimulate macrophages and synovial cells to haveinflammatory cytokines (TNF-α, IFN-γ, GM-CSF, IL-2, IL-6) and matrixmetalloproteinase secreted, for which signals were transmitted bysoluble materials such as interferon-gamma (IFN-γ) and IL-17 and by cellsurface component such as CD69. The secreted cytokines stimulate theproliferation of synovial membrane to form a pannus and destroycartilage in cooperation with matrix metalloproteinase. The activatedCD4+ T cells induce the activation of B cells through the contact withthem on cell surface by CD40L, CD28, and a1b2 integrin, leading to theproduction of antibody containing rheumatoid factors. When CD4+ T cellsare activated, osteoprotegerin ligand is expressed on the surface, whichstimulates osteoclastogenesis, an important factor for bone destruction(Kong Y Y, Feige U, Sarosi I., et al.: Activated T cells regulate boneloss and joint destruction in adjuvant arthritis through osteoprotegerinligand. Nature 402, 304-309, 1999). The activated macrophages andfibroblasts accelerate angiogenesis by secreting VEGF, FGF, etc. Theactivated vascular endothelial cells in synovial membrane make anamplified cycle of inflammation by secreting chemokine such as IL-8,inducing the expression of adhesion molecule and speeding up theinfiltration of inflammatory cells. Rheumatoid arthritis is alsobelieved to be a T-cell mediated autoimmune disease, which is related tothe antigen-nonspecific intracellular interaction between T-lymphocytesand antigen-presenting cells. The reaction size of T-cells is determinedby simultaneous stimuli induced by the interaction between a T-cellsurface molecule and its' ligand. A major simultaneous stimulus signalis given by the interaction between T-cell surface receptors, CD28 andCTLA4, and their ligands such as B7-related molecules onantigen-presenting cells, that is CD80 (B7-1) and CD86 (B7-2) (Linsley,P. and Ledbetter, J.: The role of the CD28 receptor during T cellresponses to antigen. Ann. Rev. Immunol. 11:191-212, 1993). T-cellactivation without simultaneous stimuli results in anergic T-cellresponse, indicating that immune system does not response to a stimulus[Schwartz, R. H.: Costimulation of T lymphocytes: the role of CD28,CTLA-4, and B7/BB1 in interleukin-2 production and immunotherapy. Cell71:1065-1068, 1992].

Fundamental treatment of arthritis to cure the cause is still far, andall the medicines developed so far are just for relieving a pain,inhibiting inflammation or keeping the function as it is. Such medicinesare supposed to be administered for a long time, but long-termadministration of those drugs cause side effects in gastrointestinalsystem, central nervous system, hematopoietic organ, kidney, liver, etc.(Langenegger T, Michel B A.: Drug treatment for rheumatoid arthritis.Clin. Orthop. 366:22-30, 1999).

As explained hereinbefore, arthritis related diseases are considered tobe chronic inflammatory diseases and T-cell medicated immune systemdisorders, so that it is an urgent need, for the treatment of suchdiseases, to develop a medicine to inhibit release of cytokine and todestroy activated T cells selectively.

The present inventors have made every effort to find out a material fromherb medicines that can inhibit release of cytokine and destroyactivated T-cells only. And the present inventors have completed thisinvention by confirming that Notoginseng radix extract can inhibitseparation of cytokine and destroy activated T-cells only.

DISCLOSURE OF INVENTION

Technical Solution

It is an object of the present invention to provide a compositioncomprising Notoginseng radix extract for preventing and treatingarthritis as an effective ingredient.

BRIEF DESCRIPTION OF THE DRAWINGS

The application of the preferred embodiments of the present invention isbest understood with reference to the accompanying drawings, wherein:

FIG. 1 is a schematic diagram showing the method for extracting andseparating Notoginseng radix extract of the present invention,

FIG. 2 is a graph showing the effect of Notoginseng radix extract of thepresent invention on release of tumor necrosis factor-alpha (TNF-α),

FIG. 3 is a graph showing that Notoginseng radix extract of the presentinvention destroys activated T-cells selectively,

FIG. 4 is a graph showing the inhibiting effect of Notoginseng radixextract of the present invention on the arthritis progress tested byusing animals with type 2 collagen induced arthritis, which is presentedby arthritis index,

FIG. 5 is a set of photographs showing the inhibiting effect ofNotoginseng radix extract of the present invention on the arthritisprogress tested by animals with type 2 collagen induced arthritis.

BEST MODE FOR CARRYING OUT THE INVENTION

In order to achieve the above object, the present invention provides acomposition comprising Notoginseng radix extract for preventing andtreating arthritis as an effective ingredient.

The composition of the present invention includes a pharmaceuticalcomposition for preventing and treating arthritis and a composition forhealth food

Notoginseng radix extract of the present invention inhibits release oftumor necrosis factor-alpha (TNF-α) and is the death of activatedT-cells selectively, so that it can be effectively used for theproduction of improved health food or the development of a medicine forpreventing and treating arthritis.

Notoginseng radix is a root of a perennial herb belonging to Panaxnotoginseng (Burk.) F. H. Chen. It is smaller than a ginseng and has 7pieces of leaves. Its' root is in a small thread drum shape and it israised widely in Yunnan and Sichuan, southern China. Since the plant has7 leaves on three branches, it has been called ‘Samchil (three-seven)’and often called ‘Samchil ginseng’ owing to its similar appearance toKorean ginseng. The root has 3-8% saponin and its' major components areginsenoside Rb1, Rg1 and Re, and notoginsenoside R1, R2, Fa and Fc, butsmall amount of ginsenoside R2, b2, d, e, c are also included. R0 is notcontained or if it is, it must be least. Essential oil composition isfewer in Notoginseng radix than in Panax ginseng. Notoginseng radixadditionally includes oleanolic acid. Its' root has hemostatic andcardiotonic activities. It was confirmed from animal tests that the roothas efficacy of increasing blood flow of coronary artery, decreasingoxygen consumption of cardiac muscle and lowering the levels of lipidand cholesterol in blood. Notoginseng radix also has functions ofanti-inflammation, analgesia and hemostasis, so that it is very usefulfor the treatment of not only inflammatory diseases including hepatitisbut also bleeding from trauma, cut, etc., and internal hemorrhage.Applying to a wound or oral administration give the same effects.

Notoginseng radix extract of the present invention is extracted by usingwater, alcohol or a mixed solvent of water and alcohol. At this time,alcohol is preferred to be ethanol.

Conventional extraction methods including cold precipitation, hotprecipitation, heating, etc., using the solvent mentioned above areused.

Notoginseng radix extract of the present invention inhibits release oftumor necrosis factor-alpha (TNF-α), so that it can be used for theproduction of health food or a medicine for preventing and treatingarthritis.

In order to investigate how Notoginseng radix extract of the presentinvention worked to inhibit release of tumor necrosis factor-alpha(TNF-α), THP-1 cells, a human monocytic cell line, were treated withlipopolysaccharide (LPS) and Notoginseng radix extract of the presentinvention at the concentration of 2 or 10 μl/ml. Then, the amount ofreleased tumor necrosis factor-alpha (TNF-α) in cell culture medium wasmeasured by ELISA. As a result, the amount of released tumor necrosisfactor-alpha (TNF-α) was remarkably decreased by the treatment of 10μl/ml of Notoginseng radix extract of the present invention (seeExperimental Example 1).

Notoginseng radix extract of the present invention can be used for theproduction of health food or a medicine for preventing and treatingarthritis owing to its ability to death activated T-cells selectively.

In order to investigate whether or not Notoginseng radix extract of thepresent invention was able to death activated T-cells only, a lymph nodeof a 5-week-old female mouse was taken and single cells were prepared.The cells were cultured, during which T cells were activated Theapoptosis of activated T-lymphocytes was investigated. As a result, whencells were treated with over 5 μl/ml of Notoginseng radix extract of thepresent invention, only activated T-cells were killed (inactivatedT-cells were still alive) (see Experimental Example 2).

Notoginseng radix extract of the present invention also inhibits theprogress of the disease in animals having type 2 collagen inducedarthritis.

In order to investigate the treatment effect on arthritis of Notoginsengradix extract of the present invention, collagen suspension wasintra-dermally injected in tail head of a mouse to induce arthritis.Notoginseng radix extract of the present invention was orallyadministered to the mouse with arthritis, which was then observed. As aresult, the progress of arthritis was remarkably inhibited from the9^(th) day after oral administration of the extract (see ExperimentalExample 3).

A composition of the present invention can additionally include, inaddition to Notoginseng radix extract, one or more effective ingredientshaving a similar to or the same function as Notoginseng radix extract.

A composition of the present invention can additionally include, inaddition to Notoginseng radix extract, one or more effective ingredientshaving a different function from that of Notoginseng radix extract.

A composition of the present invention can contain at least one ofpharmaceutically acceptable carriers, in addition to the above effectiveingredients, for the convenience of the administration. Pharmaceuticallyacceptable carriers can be selected from a group consisting of saline,sterile water, Ringer's solution, buffered saline, dextrose solution,maltodextrin solution, glycerol, ethanol and a mixture of them (one ormore components). If necessary, other additives such as anti-oxidants,buffers, fungistats, etc., can be included. A composition of the presentinvention can also be prepared in the forms of pills, capsules,granules, tablets and injectable solutions such as acqueous solutions,suspensions, emulsions, etc., produced by being mixed with generallyused diluents, disintegrating agents, surfactants, binders andlubricants. Besides, a composition of the present invention can beprepared in different forms considering a disease and includedingredients by general method well-known to the people in this field orthe method described in Remington's Pharmaceutical science (Newestedition), Mack Publishing Company, Easton Pa. Calcium or vitamin D₃ canbe added to a composition of the present invention to enhance itsmedicinal effect of preventing and treating arthritis.

The administration method of a composition of the present inventionvaries from the purpose of the treatment; either oral administration orparenteral administration (for example, intravenous, intradermal,intraperitoneal or local injection) is fine. And the dosage of thecomposition is determined awarding to weight, age, gender, healthcondition of a patient, diet, administration times and method, excretionrate, and severity of a disease. The effective usage of Notoginsengradix extract of the present invention is 0.1˜10 mg/kg, and 0.1˜3 mg/kgis more preferable. The administration times can be once a day orpreferably several times a day.

The acute toxicity test in mice via oral administration was performed tosee if the Notoginseng radix extract of the present invention has acutetoxicity in mice. As a result, its estimated LD₅₀ values are muchgreater than 2 g/kg in mice, indicating that this extract is evaluatedto be a safe substance.

A composition of the present invention can be treated for preventing andtreating arthritis either independently or in combination with surgicaloperation, radiotherapy, hormone therapy, chemotherapy and otherbiological response regulators.

A composition of the present invention can be added to health food toimprove arthritis related diseases. Notoginseng radix extract of thepresent invention can be added to food as it is or together with otherfood or food ingredients by general method for food process. The mixingratio of effective ingredients is determined by the purpose of use (forprevention, for promoting health, or for treatment of a disease). Ingeneral, Notoginseng radix extract of the present invention is added tofood or beverages under 100 weight %, preferably under 50 weight %.However, in the case of long-term administration for the purpose ofhealth and sanitation or health control, the amount of a compositionadded to food or beverages might be less than the above, but since thecomposition is safe for human, it could be added more than the above.

There is no limitation in food category applicable to the extract of thepresent invention. So, the extract can be added to meat, sausages,bread, chocolate, candies, snacks, cookies, pizza, ramyun, noodles,gums, dairy product including ice cream, soups, beverages, tea, drinks,alcoholic drinks and vitamin complex, etc. and other ordinary healthfood.

A composition for health promoting beverages can additionally includevarious flavors or natural carbohydrates, like any other ordinarybeverages. Natural carbohydrates are exemplified by monosaccharides suchas glucose and fructose, disaccharides such as maltose and sucrose,polysaccharides such as dextrin, cyclodextrin, and sugar alcohols suchas xilytole, sorbitol and erythritol. As a sweetening agent, naturalsweeteners such as thaumatin and stevia extract, and syntheticsweeteners such as saccharin and aspartame can be used. It is preferredto add natural carbohydrates by 0.1˜20 g per 100 ml of a composition ofthe present invention, and is more preferred to add 1˜10 g of naturalcarbohydrates to 100 ml of the composition.

In addition to the above, a composition of the present invention canalso include various nutrients, vitamins, electrolytes, flavoringagents, coloring agents, pectic acid and its salts, alginic acid and itssalts, organic acids, protective colloidal thickeners, pH regulators,stabilizers, antiseptics, glycerin, alcohol, carbonating agents used incarbonated beverages, etc. The composition of the present invention canfurther include sarcocarps to produce natural fruit juices, fruitbeverages and vegetable beverages. Each ingredient is used eitherindependently or in combination with others. At this time, the mixingrate is not so important but in general, 0.05˜50 parts of weight per 100parts of weight of the composition of the present invention ispreferred.

MODE FOR THE INVENTION EXAMPLES

Practical and presently preferred embodiments of the present inventionare illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, onconsideration of this disclosure, may make modifications andimprovements within the spirit and scope of the present invention.

Example 1 Preparation of Notoginseng radix Extract

Cultivated Notoginseng radix was purchased from a wholesale driedmedicinal herb store.

<1-1> Preparation of Notoginseng radix Crude Extract

<1-1-1> Crude Alcohol Extract of Notoginseng radix

Notoginseng radix was cut into 1˜2 cm fragments. The fragments werewashed with running water to eliminate impurities. The fragments werepulverized. 200 g of the Notoginseng radix powder was put in a 3 lflask, which was stirred at reflux at 78.5° C. using 2,000 ml ofethanol. Extraction by heating was repeated three times for 4 hours. Theextract was filtered and vacuum-concentrated under reduced pressure byusing vacuum rotary evaporator under 40° C., resulting in Notoginsengradix crude extract containing 2.7 g of Notoginseng radix powder (RF1M)(yield: 1.35%).

<1-1-2> Crude Water Extract of Notoginseng radix

Notoginseng radix crude extract was extracted by the same method asdescribed in the above <1-1-1> and the only difference in the procedurewas that water was used instead of ethanol as an extraction solvent.

<1-1-3> Crude Mixed Solvent Extract of Notoginseng radix

Notoginseng radix crude extract was extracted by the same method asdescribed in the above <1-1-1> and the only difference in the procedurewas that a mixed solvent of water (25%) and ethanol (75%) was usedinstead of ethanol as an extraction solvent.

<1-2> Separation of Notoginseng radix Crude Extract

A fraction (RF1MB) was obtained from the crude extract (FF1M) preparedin the above <1-1-1> at room temperature by using 500 ml of normalbutanol (n-butanol) as a solvent, for which a fraction funnel was usedand solvent fractionation was repeated three times.

RF1MB4 fraction was separated from the RF1MB fraction by columnchromatography. Column chromatography was performed again with theRF1MB4 fraction, resulting in the final fraction of Notoginseng radixextract (RF1MB4b).

Extraction and separation method of Notoginseng radix extract of thepresent invention is described in FIG. 1.

In experimental examples of the invention, the final extraction ofNotoginseng radix extract (RF1MB4b) was concentrated and thenfreeze-dried. The dried fraction was diluted with water and used for invitro and animal tests.

Experimental Example 1 Inhibition of the Release of TNF-α by Notoginsengradix Extract of the Present Invention

Following experiments were performed to investigate whether or notNotoginseng radix extract of the present invention inhibited the releaseof TNF-α, a cytokine separated from human monocytic cell line ‘THP-1cell’.

<1-1> Cell Selection and Culture

The below cell line was used to investigate the effect of Notoginsengradix extract of the present invention on the release of TNF-α.

Human originated cell line THP-1 (ATCC No. TIB-202) was purchased fromATCC (Rockville, USA) and cultured in RPMI 1640 (Gibco, BRL, USA) mediumsupplemented with 10% FBS (fetal bovine serum).

<1-2> Quantification of released TNF-α

In order to investigate the effect of Notoginseng radix extract of thepresent invention on the release of TNF-α, the amount of released TNF-αwas measured by ELISA using cells prepared in the above <1-1>.

Cells were plated into a 96-well plate by 5×10⁵ cells/ml andlipopolysaccharide (LPS) was added in order to activate cells for therelease of TNF-α.

An experimental group was treated with Notoginseng radix extract(RF1MB4b) at the concentration of 2 or 10 μl/ml together with LPS. Afterthe treatment, the released TNF-α in culture supernatant was quantifiedby ELISA.

The results are presented in FIG. 2.

As shown in FIG. 2, when an experimental group was treated with lowconcentration (2 μl/ml) of Notoginseng radix extract (RF1MB4b), theamount of released TNF-α of the experimental group was just a littledifferent from that of a control group not treated with the extract.But, when the extract was provided with high concentration (10 μl/ml),the amount of released TNF-α in the experimental group was greatlydecreased, comparing to a control group.

Thus, the above results indicate that Notoginseng radix extract of thepresent invention inhibits the release of TNF-α.

Experimental Example 2 Selective Apoptosis of Activated T-Cells byNotoginseng radix Extract of the Present Invention

In order to confirm whether or not Notoginseng radix extract of thepresent invention could destroy activated T-cells only, followingexperiments were performed.

<2-1> Separation and Activation of T-Cells

A lymph node of a 5-week-old female mouse was taken out and mashed bythe back tip of a sterilized syringe to extract cells. The cells werefiltered by a cell-filter (Falcon, NJ USA) and washed with PBS, then putin a culture medium at the concentration of 2×10⁶cells/ml. As a culturemedium, RPMI 1640 (Gibco, BRL, USA) supplemented with 10% FBS (fetalbovine serum) was used.

In order to activate T-cells only, concanavalin A was added by 5 μg/mlto the medium, followed by culture for 48 hours. After 48 hours ofculture, 10 mg/ml of methyl-α-D-mannopyranoside (sigma, Germany) was putin the medium, followed by further culture for 30 minutes. Then, thecells were washed with PBS three times and put in a culture mediumsupplemented with 100 units/ml of human interleukine-2 (hIL-2, R&D, MN,USA), followed by further culture for 48 hours and cell density wasmaintained as 2×10⁶ cells/mg during the culture (Lenardo M J. et al.:Interleukin-2 programs mouse alpha beta T lymphocytes for apoptosis.Nature. 353(6347):858-61. 1991).

<2-2> Investigation of Selective Apoptosis of Activated T-Cells

The concentration of activated T-cells was adjusted to 1×10⁶ cells/ml,then they were put in a 96-well plate (Falcon, USA) by 200 μl/well. Atthat time, 100 units/ml of human interleukine-2 (hIL-2) was added toeach well.

While a control group was not treated with Notoginseng radix extract, anexperimental group was treated with the final fraction (RF1MB4b) ofNotoginseng radix extract prepared in the above example at differentconcentrations (5 μg/ml, 10 μg/ml, 20 μg/ml) before being cultured for24 hours.

As a control, inactivated cells were prepared as follows.

Single cells were collected from spleen and cell density was adjusted to2×10⁶ cells/ml, which were distributed to a 96 well plate by 200μl/well. Notoginseng radix extract was added thereto, followed byculture for 24 hours. After 24 hours of culture, the cells weretransferred to a flow tube, to which propidium iodide (PI) was added.Then, live cells were counted for 20 seconds by using CellQuest programof FACSCaliver (Becton Dickinson, France).

Apoptosis was calculated as follows: (1−F extract treatedcells/untreated cells)×100. All candidate drugs were examined by thatmath formula to choose a drug to induce high apoptosis of activatedT-cells but low apoptosis of naive T-cells (Sabapathy K, Hu Y, KallunkiT, Schreiber M, David J P, Jochum W, Wagner E F, Karin M.: JNK2 isrequired for efficient T-cell activation and apoptosis but not fornormal lymphocyte development. Curr. Biol. 11;9(3): 116-25. 1999).

The results are presented in FIG. 3.

As shown in FIG. 3, when Notoginseng radix extract of the presentinvention was treated with high concentration over 5 μl/ml, activatedT-cells were selectively destroyed while inactivated T-cells stillremained.

Thus, it was confirmed that Notoginseng radix extract of the presentinvention destroys activated T-cells selectively and the apoptosiseffect was concentration-dependent.

Experimental Example 3 Inhibition of the Progress of Arthritis in TestAnimals with Type 2 Collagen Induced Arthritis by Notoginseng radixExtract of the Present Invention

In order to investigate whether or not Notoginseng radix extract of thepresent invention could inhibit the progress of arthritis in testanimals having type 2 collagen induced arthritis, following experimentswere performed.

<3-1> Inducement of Arthritis in Test Animals

In order to prepare test animals having type 2 collagen inducedarthritis, 5-6 week old male DBA1 mice were purchased from SCI company,Japan, and the mice were raised at 21° C. with 40% humidity.

Bovine type 2 collagen (Condrex Co., Japan) was dissolved in 0.05%acetic acid, making the concentration 2 mg/ml. Then the type 2 collagenwas mixed with the same amount of complete adjuvant (Condrex Co.,Japan). While cooling down with ice, the mixture became homogeneoussuspension by using T-connector linked to 3 ml syringe. After confirmingthe suspension was prepared rightly, tail head of a mouse was sterilizedwith alcohol cotton and 100 μl of collagen suspension was injected underthe skin of the tail head.

<3-2> Oral Administration of Notoginseng radix Extract (RF1MB4b) of thePresent Invention

Notoginseng radix extract (RF1MB4b) prepared in the above example wasdissolved in water, resulting in 2.5 mg/ml solution. The solution wasfiltered by 0.25 uM filter.

The filtered solution was diluted to 0.2 mg/ml and was administered tothe mouth of a mouse through sonde linked to a 1 ml syringe, once a dayand by 0.05 mg/250 μl/mouse.

<3-3> Progress of Arthritis: Naked Eye Observation and Diagnosis

In order to investigate arthritis treating effect of Notoginseng radixextract (RF1MB4b) of the present invention, the Notoginseng radixextract (RF1MB4b) prepared in the above example was administered by thesame method as described in the above <3-2> to test animals havingarthritis induced by the injection of collagen suspension.

Arthritis was developed 30 days after collagen suspension was injectedto a mouse. Naked eye observation on lesion of arthritis was performedby using following scores based on literature cited.

0: No swelling or flair, 1: Light swelling and flair in joint, 2: Clearswelling and flair in joint, 3: Severe swelling and flair in jointincluding knuckle joint, 4: Severe swelling in all over the joint.

Therefore, the highest score of lesion of arthritis is 16 per mouse,which sums up scores of forelegs and hind legs, and the highest scoreper one leg is 4 (Courtenay J S, Dallman M J, Dayan A D, et al.:Immunization against heterologous type II collagen induces arthritis inmice. Nature 283: 666-668. 1980).

FIG. 4 and FIG. 5 present the results of investigation, after oraladministration of the extract, of arthritis progress inhibiting effectof Notoginseng radix extract of the present invention in test animalswith type 2 collagen induced arthritis.

In FIG. 4, the arthritis progress inhibiting effect of Notoginseng radixextract of the present invention in test animals with type 2 collageninduced arthritis was presented as arthritis index, and FIG. 5 is a setof photographs showing the arthritis progress inhibiting effect ofNotoginseng radix extract of the present invention in animals havingtype 2 collagen induced arthritis.

As shown in FIG. 4, when Notoginseng radix extract of the presentinvention was orally administered into a mouse having type 2 collageninduced arthritis, the progress of the disease was obviously inhibitedfrom the 9^(th) day of administration, comparing to a control group.

As shown in FIG. 5, both a control medicine without Notoginseng radixextract and an experimental medicine including the extract were orallyadministered respectively to mice having type 2 collagen inducedarthritis. Big difference between the two was observed after 21 daysfrom the administration. A mouse treated with a control medicine showedvery severe swelling all over the joints but a mouse administered withan experimental medicine just showed light flair and swelling in joints.

Therefore, it was confirmed that Notoginseng radix extract of thepresent invention effectively inhibits the progress of arthritis.

Example 4 Acute Toxicity Test with Notoginseng radix Extract of thePresent Invention

Notoginseng radix extract of the present invention is classified into afood material, indicating that it is safe. But, for the use as atreatment medicine, acute toxicity of the extract had to be investigatedas follows.

6-week old SPF mice were used in the tests for acute toxicity.Notoginseng radix extract (RF1MB4b) prepared in the above example wassuspended in distilled water and orally administered once to 5 mice pergroup at the dosage of 2, 1, and 0.5 g/kg.

Death, clinical symptoms, and weight change in mice were observed,hematological tests and biochemical tests of blood were performed, andany abnormal signs in the gastrointestinal organs of chest and abdomenwere checked with eyes during autopsy.

The results showed that Notoginseng radix extract of the presentinvention did not cause any specific clinical symptoms, weight change,or death in mice. No change was observed in hematological tests,biochemical tests of blood, and autopsy.

Notoginseng radix extract (RF1MB4b) of the present invention used inthis experiment is evaluated to be safe substance since it does notcause any toxic change in mice up to the level of 2 g/kg and itsestimated LD₅₀ values are much greater than 2 g/kg in mice.

Manufacturing Example 1 Preparation of Pharmaceutical Formulations

<1-1> Preparation of Powders

Notoginseng radix extract 2 g

Lactose 1 g

Powders were prepared by mixing all the above components and filledairtight bag with them.

<1-2> Preparation of Tablets

Notoginseng radix extract 100 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

Tablets were prepared by mixing all the above components by theconventional method for preparing tablets.

<1-3> Preparation of Capsules

Notoginseng radix extract 100 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

Capsules were prepared by mixing the components above and filled gelatincapsules with them according to the conventional method for capsules.

Manufacturing Example 2 Preparation of Food

Foodstuff containing Notoginseng radix extract of the present inventionwas prepared as follows.

<2-1> Preparation of Cooking Spices

Health improving spices and condiments containing Notoginseng radixextract of the present invention by 20-95 weight % were prepared.

<2-2> Preparation of Tomato Ketchup and Sauce

Health improving tomato ketchup or sauce was prepared by addingNotoginseng radix extract of the present invention by 0.2-1.0 weight %to original tomato ketchup or sauce.

<2-3> Preparation of Flour Food

Health improving flour food was prepared by adding Notoginseng radixextract of the present invention by 0.5-5.0 weight % to wheat flour andthen making the flour into bread, cakes, cookies, crackers and noodles.

<2-4> Preparation of Soups and Gravies

Notoginseng radix extract of the present invention was added by 0.1-5.0weight % to soups and gravies to produce health improving processedmeats, noodle soups and gravies.

<2-5> Preparation of Ground Beef

Notoginseng radix extract of the present invention was added by 10weight % to ground beef to prepare health improving ground beef.

<2-6> Preparation of Dairy Products

Notoginseng radix extract of the present invention was added by 5-10weight % to milk to prepare health improving dairy products such asbutter, ice cream, etc.

<2-7> Preparation of Sunsik

Brown rice, barley, glutinous rice and coix (job's tear) weregelatinizated by the conventional method, followed by drying. The driedmixture was distributed and pulverized, resulting in 60-mesh grain sizeof powders.

Black bean, black sesame and perilla were steamed and dried by theconventional method. The dried mixture was distributed and pulverized,resulting in 60-mesh grain size of powders.

Notoginseng radix extract of the present invention wasvacuum-concentrated under reduced pressure using a vacuum concentrator,which was then spray-dried with a hot-air drier. The dried material waspulverized by a grinder, resulting in 60-mesh grain size of powders.

The prepared grain, seeds, and dried Notoginseng radix extract powderswere all mixed at the following ratio.

Grain (brown rice 30 weight %, coix 15 weight %, barley 20 weight %),

Seeds (perilla 7 weight %, black bean 8 weight %, black sesame 7 weight%),

Dried powder of Notoginseng radix extract (3 weight %),

Ganoderma lucidum (0.5 weight %),

Rehmannia glutinosa (0.5 weight %)

Manufacturing Example 3 Preparation of Beverages

<1-1> Preparation of Carbonated Beverages

Sugar (5-10%), citric acid (0.05-0.3%), caramel (0.005-0.02%) andvitamin C (0.1-1%) were mixed, to which purified water (79-94%) wasadded to make syrup. The prepared syrup was sterilized at 85-98° C. for20-180 seconds, then mixed with cooling water at the ratio of 1:4. Then,carbon dioxide gas (0.5-0.82%) was given to the mixture to preparecarbonated beverages containing Notoginseng radix extract of the presentinvention.

<1-2> Preparation of Health Beverages

Acid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%) andwater (75%) were all mixed with Notoginseng radix extract evenly,followed by sterilization. The mixture was put in a small container suchas a glass bottle or pat bottle, resulting in health beverages.

<1-3> Preparation of Vegetable Juice

5 g of Notoginseng radix extract of the present invention was added to1,000 ml of tomato or carrot juice to prepare health vegetable juice.

<1-4> Preparation of Fruit Juice

1 g of Notoginseng radix extract of the present invention was added to1,000 ml of apple or grape juice to produce health fruit juice.

INDUSTRIAL APPLICABILITY

As explained hereinbefore, Notoginseng radix extract of the presentinvention has activities of inhibiting TNF-α release and destroyingactivated T-cells selectively.

Therefore, Notoginseng radix extract of the present invention can beeffectively used for the production of health food or a medicine forpreventing and treating arthritis.

1. A composition comprising Notoginseng radix extract for preventing andtreating arthritis as an effective ingredient.
 2. The composition forpreventing and treating arthritis as set forth in claim 1, wherein theNotoginseng radix extract is extracted by water, alcohol or a mixedsolvent of water and alcohol.
 3. The composition for preventing andtreating arthritis as set forth in claim 1, wherein the alcohol isethanol.
 4. A pharmaceutical composition for preventing and treatingarthritis comprising the composition of anyone of claim 1 to claim
 3. 5.A health food composition for preventing and treating arthritiscomprising the composition of anyone of claim 1 to claim 3.